Detection and identification of acid-fast bacilli of Mycobacterium species by conventional procedures requires growing the organisms from patient specimens and then testing the isolates for various phenotypic characteristics. These methods may take days to 1 or more months. The development of a few highly specific molecular probes for testing cultures growing acid-fast bacilli has greatly reduced the time to identification of some mycobacterial isolates. Recently, the polymerase chain reaction and isothermal nucleic acid amplification techniques have been used in assays that offer a high degree of specificity and reasonable sensitivity for detection of Mycobacterium tuberculosis in clinical samples. It would be useful to have amplification assay systems that are capable of detecting multiple Mycobacterium species while excluding cross-reactive signals from other bacteria commonly present in clinical samples. Experiments have been successfully performed with an assay version that simultaneously detects the presence or absence of nucleic acid amplification products from six common clinically isolated Mycobacterium species. A joint patent application between the N.I.H. and Beckman Coulter Corporation has been recently submitted. Because sample preparation is a critical component of molecular diagnostic assays, we have initiated a new effort to investigate the usefulness of a pressure cycling technology (PCT) for improving the efficiency of nucleic acid release from mycobacterium cells. A barocycler (device for pressure cycling) has been recently brought into CPD under a CRADA with BBI Biotech. Using PCT as a pretreatment before nucleic acid extraction we were able to increase the average PCR signals from suspensions containing Mycobacterium gordonae and suspensions of Mycobacterium tuberculosis. A collaborative prospective study is being initiated with the Johns Hopkins Center for TB Research and BBI to evaluate the utility of different PCT conditions for respiratory specimen processing before mycobacterium detection by culture or molecular testing.